Clone h129

For surface staining, splenocytes and isolated immune cells from spleen, muscle, and lymph node tissues were stained for 15 min at room temperature with antibodies against the following proteins: CD4 (clone GK1.5, 863 eBioscience; clone H129.19, BioLegend, CA, USA), CD8 (clone 53-6.7, BD Biosciences; clone 53-6.7, Invitrogen, CA, USA), CD44 (clone IM7, Invitrogen), CD62L (clone MEL 14, BD Biosciences), CD11b (clone M1/70, Bio Legend), F4/80 (clone BM8, Invitrogen), CD86 (clone GL1, BD Biosciences), and CD11c (clone N48, eBioscience). Cells were fixed with 1% paraformaldehyde, analyzed using a FACS Canto II flow cytometer (BD Biosciences, NJ, USA), and the data were analyzed using FlowJo software (TreeStar, OR, USA).

Per mouse, 50 µL EDTA blood was diluted in 50 µL PBS and overlaid on 100 µL lymphocyte separation medium (1077, PromoCell). After centrifugation, cells were isolated from the interphase, washed and stained for 15 min at 4 °C with the following antibodies: PECy5 anti-CD4 (1:1000, clone H129.19, BioLegend), PECy7 anti-CD8 (1:500, clone 53-6.7, BioLegend), BV510 anti-B220 (1:333, clone RA3-6B2, BioLegend), PerCpCy5.5 anti-CD11b (1:1000, clone M1/70, BioLegend), PE anti-Gr1 (1:1000, clone RB6-8C5, BioLegend), and FITC anti-F4/80 (1:1000, BM8, BioLegend). After staining, cells were washed, suspended in 250 µL PBS containing 2% bovine serum albumin (#8076.3, Roth), and filtered through 40 µm cell strainers. Samples were measured on a FACSAria Sorp (BD). Total cell numbers were determined using forward and side scatter. Frequency of helper T cells (CD4+, CD8−), cytotoxic T cells (CD8+, CD4−), B cells (B220+), macrophages (CD11b+, F4/80+) and neutrophils/monocytes (CD11b+, Gr1+) were determined as percentage of total lymphocytes.